穿心莲内酯对人皮肤基底细胞癌A431细胞生长、凋亡及增值细胞核抗原蛋白表达的影响

目的 探讨穿心莲内酯（AD）对人皮肤基底细胞癌A431细胞生长、凋亡及增殖细胞核抗原（PCNA）蛋白表达的影响。方法 应用酸性磷酸酶（APA）法检测细胞增殖抑制率，荧光显微镜观察细胞形态，Annexin V-FITC/PI双染法、流式细胞术（FCM）检测细胞凋亡率， Rh123染色FCM检测细胞线粒体膜电位，免疫细胞化学法检测细胞PCNA蛋白表达。结果AD呈时间、剂量依赖性抑制A431细胞生长增殖;且同一时间点50mg/L、100mg/L AD组与溶媒对照组相比差异显著（P＜0.05）。AD组作用A431细胞24h时，部分细胞核染色质出现典型凋亡形态学改变。不同浓度AD作用A431细胞24h，早期凋亡率、晚期凋亡及坏死率均随AD浓度升高而逐渐增加，且50mg/L、100mg/L AD组与溶媒对照组相比差异显著（P＜0.05）。AD作用A431 24h细胞内PCNA蛋白随AD浓度升高表达逐渐减少。AD作用A431细胞24h后，线粒体膜电位下降明显，与溶媒对照组相比较，50mg/L、100mg/L AD组差异有统计学意义（P＜0.05）。结论 AD可明显抑制A431细胞生长增殖，其抑制细胞增殖可能与下调PCNA蛋白表达有关。AD能诱导A431细胞凋亡，其凋亡机制可能与细胞线粒体膜电位下降有关。

Effect of andrographolide on cell growth, apoptosis and expression of proliferating cell nuclear antigen protein in the human skin carcinoma A431 cell line

Objective To study influence of andrographolide（AD）on cell growth、apoptosis and proliferating cell nuclear antigen(PCNA) protein expression in human skin carcinoma A431 cells. Methods Acid phosphatase assay (APA) was used to detect
inhibition of cell proliferation. Cell morphology was observed by fluorescence microscopy. Annexin V-FITC/PI double staining method was used to detect A431 cell apoptosis by the flow cytometry (FCM). Rh123 staining detected mitochondrial membrane potential by FCM. PCFNA protein expression on A431 cells was detected by immunocytochemistry. Results AD inhibited A431 cell proliferation by time and dose dependent. There was a significant difference between 50mg/L, 100mg/L AD group and vehicle control group (P<0.05) on the same time. A part of nuclear chromatin appeared typical apoptosis morphological changes after AD group acted A431 cells for 24 hours.The early apoptosis ,late apoptosis and necrosis rates were increased with various AD concentrations treatment for 24 hours. There was a significant inhibition rate difference between 50mg/L, 100mg/L and vehicle control group(P<0.05). PCNA protein expression intensity was decreased gradually with AD treatment for 24 hours.
Mitochondrial membrane potential decreased significantly after AD acted on A431 cells for 24 hours; 50mg/L and 100mg/L AD groups had significant difference compared to vehicle control group（P＜0.05）. Conclusion AD significantly inhibits A431 cells proliferation and AD inhibiting cells proliferation may be associated with PCNA protein down-regulation.AD induces A431 cells apoptosis and AD inducing apoptosis mechanism may be related to decreasing mitochondrial membrane potential.